Project Summary/ Abstract Actions of estrogens mediated by estrogen receptor alpha (ER?) influence glucose homeostasis and vascular health. However, the molecular underpinnings of these actions, and how they can be selectively leveraged for therapeutic gain remain unknown. In recent studies we surprisingly found that ER? deletion exclusively from endothelium in mice fully negates the antidiabetogenic actions of estradiol (E2). We also found that elevating the cholesterol metabolite 27-hydroxycholesterol (27HC), which we previously discovered is an endogenous ER ligand, causes glucose intolerance in mice. Both these diabetic phenotypes were due to impaired skeletal muscle glucose disposal related to blunted muscle insulin delivery. In prior studies of atherosclerosis, we determined that whereas E2 is atheroprotective, an exaggerated increase in 27HC in hypercholesterolemic mice worsens lesion development. The Overall Goal of the proposed research in mice and cell culture is to determine HOW endothelial ER? modulation by estrogens and 27HC influences metabolic and vascular health. Aim 1 is to determine how endothelial ER? liganding by E2 versus 27HC influences glucose homeostasis. Muscle insulin delivery is governed by insulin-induced microvascular recruitment and perfusion, and insulin transcytosis from the circulation to the myocytes across the endothelial monolayer. Having now revealed that E2 stimulates and 27HC blunts insulin transcytosis by cultured endothelium, we will elucidate how a newly- identified ER? interacting protein, the small GTPase septin 11, partners with ER? to influence endothelial insulin transport. How E2 versus 27HC impacts gene expression in muscle microvascular endothelium in vivo will be determined by RNA-seq on ribosome-associated RNA. Effects of the two ER ligands on microvascular responses to insulin in muscle will be interrogated in vivo using contrast-enhanced ultrasound. Aim 2 is to determine how endothelial ER? liganding by E2 versus 27HC influences vascular inflammation and atherosclerosis. In cultured endothelium we previously showed that NF-kB activation is decreased by E2 and increased by 27HC via ER?. Having found that the latter process requires septin 11, we will discern how septin 11 mediates ER? regulation of NF-kB. How E2 and 27HC alter gene expression in the aortic endothelium in vivo will be interrogated by RNA-seq on ribosome-associated RNA. In Aim 3, recognizing that 27HC increases with either western diet intake or hypercholesterolemia, we will determine how lowering endogenous 27HC impacts the glucose intolerance and atherosclerosis that occur under these conditions, respectively. Mice will receive an inhibitor of the 27HC synthesizing enzyme Cyp27a1, or an adeno-associated virus allowing overexpression of the 27HC-metabolizing enzyme Cyp7b1. Floxed Cyp27a1 mice will be used to determine if macrophages or endothelial cells are key sources of endogenous 27HC contributing to these disorders. The proposed work will reveal how endothelial ER? serves as a critical point of convergence of the actions of endogenous ER ligands, namely estrogens and 27HC, to influence metabolic and vascular health.